JP2000143698A - Gramicidin analogue cyclic peptide and scavenger of hydrophobic organic compound using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new compound useful as a scavenger for chemical substances capable of selectively capturing specific chemical seeds by modifying a part of a chemical structure of a compound having ion-capturing ability. SOLUTION: This cyclic peptide is represented by the formula [R1 and R2 are each a group having hydrophilic group (e.g. carboxyl group) or the like; X1 to X4 are each a hydrophobic group (e.g. alkyl or aralkyl); Z1 and Z2 are each a fluorescent group (preferably an aryl); (n) and (m) are each 1-3] and at least one amino acid unit is non-natural type amino acid unit. This cyclic peptide can be synthesized by a well-known peptide synthetic method, preferably a solid phase method using gramicidin S as a model compound. The cyclic peptide can be used as a scavenger by binding to a carrier and a environmental hormone, especially phthalate is preferable as a hydrophobic organic compound. Thereby, even an highly diluted environmental hormone can be captured in high efficiency.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a gramicidin S wherein at least one amino acid is a non-natural amino acid.
The present invention relates to an analogous cyclic peptide or a salt thereof. The cyclic peptide of the present invention or a salt thereof is characterized in that it contains a hydrophobic group, a hydrophilic group and a fluorescent group. It can capture compounds and is useful as an environmental hormone capturing agent. More specifically, the present invention relates to a gramicidin S analog (cyclic peptide) into which a long-chain alkyl group that captures highly diluted environmental hormones and the like is introduced with high efficiency.
About. Therefore, the present invention provides a lipophilic organic compound using the cyclic peptide of the present invention or a salt thereof, preferably an environmental hormone capturing agent, a method for capturing and recovering the same, a method for detecting the same, and a method for detecting the same. About the kit.

[0002]

2. Description of the Related Art "Environmental hormones" have become a serious problem as chemical substances that have a great effect on ecosystems.
Endocrine disrupting chemicals (Endo
crineDisruptor)) is a substance having an action of entering the living body and disrupting its endocrine, and several dozen kinds of substances have been known so far. These environmental hormones may act as agonists or antagonists by binding to hormone receptors in the body, inhibit hormone biosynthesis, or act directly or indirectly on hormones in the blood. Are known.

[0003] Environmental hormones at issue include:
Alkylphenols such as nonylphenol, octylphenol and bisphenol A; PCBs such as PCB and p-oxyPCB; DDTs such as 2,2-bis (p-chlorophenyl) -1,1,1-trichloroethane (DDT); 3,7,4-tetrachlorodibenzo-
Examples include dioxins such as p-dioxin, phthalates such as dibutyl phthalate, di-2-ethylhexyl phthalate, and butyl benzyl phthalate, and many of these environmental hormones except organic tin compounds and the like. Are all those containing a 6-membered aromatic ring.

[0004] These environmental hormones have an action of disrupting endocrine secretion in living organisms in a very small amount. For example, nonylphenol is said to be capable of disrupting endocrine secretion in fish at a concentration of about 0.2 µg / l. , About 20-300 μg / kg of estradiol 84 pM for bisphenol A
And butylbenzyl phthalate have been reported to affect pregnancy in rats at 1 ppm drinking water. In particular, phthalates are thalidomide (N- (2,6-dioxo-
It is similar in chemical structure to the phthalimide moiety known as a teratogenic site of piperidin-4-yl) phthalimide), and teratogenic formation in a trace amount is of concern.

[0005] As described above, environmental hormones are chemical substances that have a very small amount and greatly affect ecosystems. Prevention of pollution by environmental hormones has become an urgent and serious problem in modern society. However, environmental hormones
Unlike conventional large amounts of pollutants, the presence of a trace amount is a problem, so that their recovery and removal are very difficult both technically and economically.

[0006] On the other hand, gramicidin is a peptide antibiotic produced by microorganisms, and is composed of linear gramicidin A,
In addition to the three types B and C, the following formula (II):

[0007]

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[0008] Gramicidin S which is a cyclic peptide consisting of 10 amino acids represented by the following formula is known. Gramicidin S is also a homolog of tyrosidine, and these molecules are known to form channels in biological membranes through which cations such as hydrogen ions, sodium ions, and potassium ions pass.

The present inventors have focused on the ability to capture ions of gramicidin S and the like, and by modifying a part of this chemical structure, a new chemical that can selectively capture a specific chemical species. It was believed that a material scavenger could be provided.

[0010]

SUMMARY OF THE INVENTION The present invention provides a capturing agent capable of selectively capturing a lipophilic organic compound, preferably an environmental hormone, particularly an environmental hormone containing a 6-membered aromatic ring. More specifically, as a cyclic peptide, a gramicidin analog having a plurality of hydrophobic groups (long-chain alkyl groups) in one direction of the ring, a hydrophilic group in the other direction, and also having a fluorescent group is provided. Things. The space formed by a plurality of hydrophobic groups is a place for taking in organic compounds such as environmental hormones. The present inventors have confirmed that the gramicidin analog of the present invention actually captures dibutyl phthalate, which is a kind of environmental hormone, and completed the present invention.

[0011]

The present invention provides the following formula (I):

[0012]

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(Wherein R ¹ and R ² each independently represent a group having a hydrophilic group or a group having a hydrophilic group bonded to a carrier, and X ¹ , X ² , X ³ and X ⁴ represent , Each independently represents a hydrophobic group, Z ¹ and Z ² each independently represent a fluorescent group, and n and m each independently represent 1 to 1.
Indicates an integer of 3. A) a cyclic peptide represented by:
The present invention relates to a cyclic peptide or a salt thereof, wherein at least one of the amino acid units constituting the cyclic peptide is a non-natural amino acid unit.

The present invention also relates to a lipophilic organic compound containing the cyclic peptide or a salt thereof, preferably an environmental hormone capturing agent, and a method of capturing or recovering the same. Furthermore, the present invention relates to a method for detecting a lipophilic organic compound, preferably an environmental hormone, in a medium using the cyclic peptide or a salt thereof, and a kit for detecting the same.

[0015] The cyclic peptide of the present invention is gramicidin S
Is used as a model compound.
Although the number is preferably from 18 to 18, preferably from 10 to 14, and more preferably from 10, the number of amino acid units is particularly limited as long as the ability to capture the lipophilic organic compound targeted by the present invention is not hindered. There are no restrictions. Further, the number of rings is not limited to a single ring, and may have two or more rings. Since the size of the ring formed and the number of hydrophobic groups differ depending on the increase or decrease in the number of amino acid units, consider the size, lipophilicity and affinity according to the type of lipophilic organic compound to be captured It is apparent to those skilled in the art that the number of amino acid units can be appropriately set.

The cyclic peptide of the present invention differs from its parent gramicidin S and known natural cyclic peptides in that it contains at least one unnatural amino acid. On the other hand, it is characterized in that it has a hydrophobic group, preferably a plurality of hydrophobic groups. Further, the cyclic peptide of the present invention is characterized in that the cyclic peptide has a hydrophilic group on the side opposite to the side having a hydrophobic group with respect to the ring. Further, the cyclic peptide of the present invention is characterized by having a fluorescent group.

FIG. 1 schematically shows the cyclic peptide of the present invention capturing a lipophilic organic compound. Hydrophobic part of lipophilic organic compound (1 in Fig. 1)
It is also shown that the size of the lipophilic organic compound which can be captured by the hydrophobic group (2 in FIG. 1) of the cyclic peptide (2 in FIG. 1) can be changed depending on the size of the ring of the cyclic peptide. A hydrophilic group (3 in FIG. 1) is present on the other side of the cyclic peptide, so that solubility in a hydrophilic medium such as water can be maintained. Further, by binding the portion of the hydrophilic group to a carrier, it becomes possible to use the cyclic peptide in a state of being carried on the carrier.

Reference numeral 4 in FIG. 1 denotes a probe or a reaction site, preferably a fluorescent group. The group not only makes it possible to detect the cyclic peptide of the present invention by fluorescence or the like, but also captures electrons in the lipophilic organic compound,
It preferably interacts with π electrons, for example, performs energy transfer and electron exchange, and also acts as a group for strengthening the interaction between the captured molecule and the cyclic peptide.

The hydrophilic group of the substituents R ¹ and R ² of the cyclic peptide represented by the general formula (I) is not particularly limited as long as it has hydrophilicity. For example, a carboxyl group, an amino group, a hydroxyl group or a phosphate group is preferred. The group having a hydrophilic group for the substituents R ¹ and R ² is not particularly limited as long as it is a group containing the above-described hydrophilic group, but has a large hydrophobic group on the opposite side of the ring. Therefore, it is preferable that the hydrophilic group has a group serving as a spacer so that the hydrophilic group can keep a certain distance from the ring. As such a spacer, a linear or branched alkylene group is preferable.

Accordingly, the group having a hydrophilic group of the substituents R ¹ and R ² includes a linear or branched group substituted by a hydrophilic group such as a carboxyl group, an amino group, a hydroxyl group or a phosphate group. Lower alkyl groups are preferred. Such a lower alkyl group has 1 to 10 carbon atoms, preferably 1 to 10 carbon atoms.
6, more preferably 1 to 4 linear or branched lower alkyl groups.

Examples of preferred groups having a hydrophilic group include, for example, carboxymethyl group, 2-carboxyethyl group, 3-carboxypropyl group, 4-carboxybutyl group, 5-carboxypentyl group, hydroxymethyl group, -Hydroxyethyl group, 3-hydroxypropyl group, 4-hydroxybutyl group, 5-hydroxypentyl group, aminomethyl group, 2-aminoethyl group, 3-aminopropyl group, 4-aminobutyl group, 5-aminopentyl group , A phosphomethyl group, a 2-phosphoethyl group, a 3-phosphopropyl group, a 4-phosphobutyl group, a 5-phosphopentyl group, and the like.

[0022] As radicals having more preferred hydrophilic groups have the general formula -R ³ -COOH (wherein, ^{R 3} is. To a straight or branched lower alkylene group) a group represented by Examples of a group having a more preferable hydrophilic group include a — (CH ₂ ) ₂ —COOH group.

The group having a hydrophilic group bonded to the carrier of the substituents R ¹ and R ² of the cyclic peptide represented by the general formula (I) includes the above-mentioned hydrophilic group having a hydrophilic group. And an insoluble carrier such as polystyrene and polyester.

The hydrophobic groups of the substituents X ¹ , X ² , X ³ and X ⁴ of the cyclic peptide represented by the general formula (I) are not particularly limited as long as they have hydrophobicity. Hydrocarbon groups are preferred. As the hydrocarbon group, one having 1 to carbon atoms
30, preferably 3 to 30, more preferably 3 to 30 carbon atoms.
20, more preferably 5 to 20 linear or branched alkyl groups, an aralkyl group in which the alkyl group is substituted by an aryl group described below, and a fat in which the alkyl group is substituted by an aliphatic cyclic hydrocarbon group described later. Aliphatic hydrocarbon group-substituted alkyl group, having 2 to 30 carbon atoms, preferably having 3 to 3 carbon atoms
0, more preferably 3 to 20, still more preferably 5 to 2
0 straight-chain or branched alkenyl group, an arylalkenyl group in which the alkenyl group is substituted with an aryl group described below, and an aliphatic cyclic hydrocarbon group in which the alkenyl group is substituted with an aliphatic cyclic hydrocarbon group described later. Alkenyl group, saturated or unsaturated monocyclic or polycyclic aliphatic or cyclic hydrocarbon group having 3 to 30 carbon atoms, preferably 5 to 30 carbon atoms, more preferably 5 to 20 carbon atoms, and still more preferably 5 to 10 carbon atoms. A substituted aliphatic cyclic hydrocarbon group in which the above-mentioned alkyl group or alkenyl group is substituted on the aliphatic cyclic hydrocarbon group, a C6-30, preferably C6-20, and more preferably a C6-10 group; Examples include a cyclic or polycyclic aryl group, and a substituted aryl group in which the above-mentioned alkyl group or alkenyl group is substituted on the aryl group.

These hydrophobic groups may have substituents such as chlorine and bromine. However, since the hydrophobicity decreases as the polarity increases, a substituent having a large polarity is not preferred. Preferred hydrophobic groups include long-chain alkyl groups such as butyl, hexyl, octyl and decyl. Here, the long-chain alkyl group has 3 carbon atoms.
-30, preferably 4-30 carbon atoms, more preferably 4 carbon atoms
To 20 and more preferably 5 to 20 linear or branched alkyl groups. Other preferred hydrophobic groups include
Examples include a phenyl group, a naphthyl group, a benzyl group, and a phenylethyl group.

The fluorescent groups for the substituents Z ¹ and Z ² of the cyclic peptide represented by the above general formula (I) may be any groups which absorb in the ultraviolet or far ultraviolet and have a fluorescent action. A group having an electron action such as charge transfer on a membered aromatic ring is preferred. Preferred fluorescent groups include those having 6 carbon atoms.
~ 30, preferably 6-20, more preferably 10-2
0 is an aryl group having at least one 6-membered aromatic ring, wherein one or more carbon atoms forming a ring of the aryl group may be substituted with a nitrogen atom, an oxygen atom or a sulfur atom. No. These aryl groups may be monocyclic, condensed cyclic, or polycyclic, and may have a ring-like π-electron system. It may be linked polycyclic. Further, these aryl groups may be alkyl groups,
It may be substituted with a substituent such as a hydroxyl group, an amino group, a carboxyl group, a halogen or a nitro group.

Specific examples of preferred fluorescent groups include naphthalene group, anthracene group, quinoline group, isoquinoline group, indole group and the like.

N or m of the cyclic peptide represented by the general formula (I) is an integer of 0 to 3, but as described above, the cyclic peptide of the present invention is not particularly limited in ring size. Instead, the size of the ring of the cyclic peptide can be appropriately determined according to the size of the lipophilic organic compound to be captured. Here, it should be understood that the values of n and m need to be defined, and are therefore merely integers from 0 to 3.

Preferred values of n or m are 1 to 3
It is an integer, more preferably 1 or 2. Numerical values of n and m
The substituent R in the amino acid unit repeated by¹, R
², X ²And X³Can be the same, but different
May be used. According to n and m in the general formula (I)
Repeat unit means that the same thing is repeated
Rather, the amino acid unit defined above repeats
It means to be done.

In a preferred cyclic peptide represented by the general formula (I), R ¹ and R ² are each represented by — (CH ₂ ) ₂ —CO
X ¹ , X ² , X ³ and X ⁴ are each independently an alkyl group, preferably a long-chain alkyl group, more preferably an n-butyl group or a non-hexyl group; ¹ and Z ² are a naphthyl group, and n and m are 1
Or a salt thereof.

Examples of the above-mentioned amino acids having a hydrophobic group include, for example, naturally occurring hydrophobic amino acids such as valine and leucine, and non-naturally occurring 2-aminomyristic acid and norleucine. Examples of the amino acid having a group having a hydrophilic group include, for example, natural or basic amino acids such as glutamic acid, aspartic acid and lysine, and unnatural 3-carboxypropyl glycine and 2-amino acid. Aminoethylglycine and the like.

The cyclic peptide represented by the general formula (I) of the present invention can be produced by a known peptide synthesis method. It is preferably produced by a solid phase method. FIG. 2 shows an outline of an example of the production method by the solid phase method. First, the amino group is replaced with N
-(9-Fluorenylmethoxycarbonyl) (Fmo
c) An amino acid protected with a group is immobilized on a resin,
The protecting group was removed with 20% piperidine in MF, and the amino acid protected with an Fmoc group (Fmoc)
-AA-OH) in butanol (HOBt) in benzotriazol-1-yl-oxy-tris (dimethylamino) phosphonium hexafluorophosphate (Bo
By reacting p) with diisopropylethylamine (DIEA), a peptide having one additional amino acid can be produced. By repeating this reaction, a peptide having a desired length can be produced.

Next, the obtained peptide is cyclized. This method includes cyclization on a resin (see FIG. 3) and liquid phase cyclization after cleavage from the resin (FIG. 4). See). 3 and 4 show the case where the ornithine residue of gramicidin S is replaced with glutamic acid and the D-phenylalanine is replaced with D-naphthylalanine for the purpose of studying the structure for convenience. ing.

The abbreviation DMF in FIGS. 3 and 4 is N, N-dimethylformamide, and HATU is O- (7-azabenzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexamide. Fluorophosphate, DI
EA indicates diisopropylethylamine, TFA indicates trifluoroacetic acid, and NMM indicates N-methylmorpholine. The non-natural amino acid used as a raw material can be produced according to a known amino acid production method.

In this specification, the abbreviations for the cyclic peptide of the present invention are used according to the method shown in FIG. 3 or FIG. That is, the one obtained by substituting the ornithine residue of gramicidin S with glutamic acid and substituting D-phenylalanine with D-naphthylalanine is abbreviated as “GS”, and the positions of valine and leucine of gramicidin S are amino acids X
What is aa is abbreviated as “GS-Xaa”.
Accordingly, the abbreviations used herein indicate the following.

"GS-Nle" is * Pro → Nle → Glu → Nle → Nap → Pro → Nle → Glu →
Nle → Nap → * “GS-Ala” is * Pro → Ala → Glu → Ala → Nap → Pro → Ala → Glu →
Ala → Nap → * “GS-Hxg” is * Pro → Hxg → Glu → Hxg → Nap → Pro → Hxg → Glu →
Hxg → Nap → * “GS-Ocg” is * Pro → Ocg → Glu → Ocg → Nap → Pro → Ocg → Glu →
Ocg → Nap → * respectively. Note that “* →...
"*" Indicates that both ends form a ring, and "Hx
"g" indicates 2-amino-octanoic acid, and "Ocg" indicates 2
-Amino-decanoic acid, and other symbols are those of ordinary amino acids.

The lipophilic organic compound that can be captured by the cyclic peptide of the present invention can be basically captured as long as it has an affinity for a hydrophobic group. Preferably, those having an interaction with the fluorescent group of the cyclic peptide of the present invention are more preferable. Examples of those having an interaction with the fluorescent group include a six-membered aromatic ring, and a lipophilic organic compound captured by affinity with a hydrophobic group has a preferred arrangement with the fluorescent group to form a lipophilic organic compound. An interaction occurs between the 6-membered aromatic ring of the above and the aryl group of the fluorescent group, and both are more strongly bound, and the capture can be completed.
The presence of the fluorescent group is not essential for capturing in the present invention.

Examples of preferred lipophilic organic compounds include:
Examples thereof include phthalic acid esters such as dibutyl phthalate, diethyl phthalate, dibenzyl phthalate, and butyl benzyl phthalate; pyrenes such as pyrenemethanol; and mono- or polyphenols such as nonyl T phenol and octyl phenol.

In order to capture highly diluted environmental hormones and the like with a small amount of a capturing agent with high efficiency, a large binding constant is required. ^{It was on} the order of ⁶ and it was confirmed that it could be captured with high efficiency. The fluorescent group is for quantifying the capture of environmental hormones and the like, and the hydrophilic group is for imparting appropriate water solubility to the peptide and for binding to the carrier substance.

FIG. 5 shows the cyclic peptide “GS-N” of the present invention.
"le" schematically shows the capture of pyrenemethanol, and the change in light absorption is shown by the fluorescence intensity.
The measurement results are obtained when the wavelength of the incident light is 285 nm and the concentration of GS-Nle is 1.0 × 10 ⁻⁷ mol. The addition of pyrenemethanol reduced the fluorescence of the naphthyl group at 325 nm, confirming that the former was bound to GS-Nle.

FIG. 6 is a plot of the fluorescence intensity at 325 nm of FIG. 5 against the amount of pyrene methanol added, in which GS-Nle specifically captures pyrene methanol.

FIG. 7 confirms that GS-Nle captures dibutyl phthalate, a kind of environmental hormone, in the same manner as in FIG.

From the results of these experiments, the following has been found. It has been confirmed that when an alkyl group having a length of at least a butyl group is introduced into X _{1 to} X ₄ of the compound I, trace organic compounds in water can be efficiently captured. This is also effective for environmental hormones such as dibutyl phthalate. It was confirmed that there was.

Since the cyclic peptide of the present invention has a hydrophilic group, it can be used as it is in a hydrophilic medium. In addition, a carrier can be bonded to the hydrophilic group of the cyclic peptide to make it insoluble before use.

When the cyclic peptide of the present invention is used as a detection reagent, the fluorescence intensity is reduced after the time that the lipophilic organic compound in the sample can be sufficiently captured by adding the cyclic peptide of the present invention to the sample has elapsed. By measuring, the lipophilic organic compound in the sample can be identified or quantified.

The measurement kit of the present invention comprises a cyclic peptide of the present invention for identifying or quantifying a sample,
As a measuring method, a conventional measuring device capable of measuring the fluorescence intensity can be used.

The gramicidin analogs according to the present invention are attracting attention as one effective and interesting means as a countermeasure against environmental hormones, which is currently a major problem. That is, the substance can be an endocrine disruptor and a detection means.

[0048]

EXAMPLES Next, the present invention will be described specifically with reference to examples, but the present invention is not limited to these examples.

Example 1 (GS-Nle, GS-Hxg,
Preparation of GS-Ocg, GS-Ala) Hereinafter, the Wang-PEG resin used is a resin having the following structure, which is commercially available, for example, from Watanabe Chemical Industry Co., Ltd. (Hiroshima).

[0050]

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Weigh 1 g of Wang-PEG resin and add
Add about 10 mL and swell for about 3 hours. This is a reaction vessel with a glass filter (with a capacity of about 12 mL)
Put in. The introduction rate described on the resin label (0.23
(mmol / g resin), and calculate the number of moles of amino acid that can be introduced into the weighed resin amount (0.23 mmol).
l). 941 mg (2.3 mmol) of Fmoc-Gl
u-OAl (equivalent to 10 equivalents) is placed in a 30 mL eggplant flask, and the minimum amount of dichloromethane (DC
Dissolve in M). Further, 0.18 mL of diisopropylcarbodiimide (DIPCDI: equivalent to 5 equivalents) is added, and the mixture is sealed and stirred at 0 ° C. for 30 minutes. When this mixture is concentrated by an evaporator, crystals are precipitated. The remaining crystals are dissolved in a minimal amount of DMF and added to the swollen resin in the reaction vessel. 28 mg of dimethylaminopyridine (DMA
Add P) as a powder, stopper the reaction vessel and gently shake for 1 hour. The reaction solution is removed by suction, and DMF (3 times)
And methanol (3 times) to wash the resin. The resin after washing with methanol is dried under reduced pressure. The dry resin yield was about 1 g. To evaluate the introduction rate of amino acids into the resin, a portion (about 5 mg) of the dried resin prepared above was accurately weighed,
Place in a 1 cm cell for UV spectrum measurement. Add 3 ml of 20% piperidine / DMF and mix well. After sinking the resin to the bottom of the cell, the absorbance at 290 nm is measured to calculate the amount of Fmoc groups released (e290 = 4950).
Is used). In the case of Fmoc-Glu (OH) -OAl, it was 0.22 mmol / g resin.

Examples 2 to 5 (Elongation of peptide chain (Fmo
c-Xaa-D-napAla-Pro-Xaa-Gl
u (OtBu) -Xaa-D-napAla-Pro-X
aa-Glu (-Wang-PEG-resin) -OAl synthesis) 230 mg of dry resin was added to a 12 mL reaction vessel, and DM was added.
Add 5 mL of F and swell for about 3 hours. The solvent is removed, and solid phase peptide synthesis is performed in each cycle according to the following procedure. 1. Add about 6 mL of 20% piperidine / DMF and shake for 10 minutes. (Removal of Fmoc group) The reaction solvent is removed by suction, and about 6 mL of DM
Wash with F three times. 3. Confirm that the result is positive in the Kaiser test described below. 4.3 equivalents of the following Fmoc amino acid and 66 m
g of Bop (3 equivalents) and 20 mg of HOBt (3 equivalents) are dissolved in DMF, 26 μL of diisopropylethylamine (DIEA) (3 equivalents) is added to the solution, and then put into the reaction vessel. Shake for 30 minutes. (Condensation) 5. The reaction solution was removed and washed three times with DMF. 6. Confirm that the result is negative in the Kaiser test described below. If positive, 4. Return to 7.1. Return to

4. The Fmoc amino acid used in the step was condensed in the following order in each cycle. I. Fmoc-Xaa-OH (Xaa = Ala) 47 mg (Xaa = Nle) 53 mg (Xaa = Hxg) 57 mg (Xaa = Ocg) 61 mg II. Fmoc-Pro-OH 51 mg III. Fmoc-D-napAla-OH 66 mg IV. Same as Fmoc-Xaa-OH 1 Fmoc-Glu (OtBu) -OH 64 mg VI. Same as Fmoc-Xaa-OH 1 VII. Fmoc-Pro-OH 51 mg VIII. Fmoc-D-napAla-OH 66 mg IX. Same as Fmoc-Xaa-OH 1

The Kaiser test will be described. First,
Prepare the following solution. Ninhydrin (5 g) / Ethanol (100 mL) Phenol (80 mL) / Ethanol (20 mL) 0.01 M KCN / H ₂ O (2 mL) / Pyridine (98 mL) A few particles of the resin were collected in a glass tube, and each of the above reagents was added to a glass tube. Add dropwise and mix well. Heating in boiling water (100 ° C.) for 2 minutes causes the resin to be colored blue and judged to be positive.

After the completion of all the cycles, a cyclization reaction is carried out. 173 mg of PdO (PPh ₃ ) ₄ was dissolved in a dissolving agent (5%
AcOH, 2.5% NMM / CHCl ₃ ) is dissolved in 5 mL. Add to the reaction vessel and shake for 2 hours to remove allyl ester. Wash three times each with washing solution (5% DIEA, 5% sodium diethyldithiocarbamate / DMF) and DMF. Add 20% piperidine / DMF and shake for 10 minutes to remove Fmoc group. Wash 3 times with DMF. Dissolve 56 mg HATU in 5 mL DMF and add
Add μL DIEA and place into reaction vessel. Shake for 30 minutes. Wash 3 times with DMF. If the above-mentioned Kaiser test is negative, the reaction is terminated.

Cleavage from the resin and removal of protecting groups.
For 225 mg of the peptide resin, a cleavage reagent (m-cresol: thioanisole: trifluoroacetic acid (TF
A) = 2: 12: 86) and shaken for 2 hours. After filtering off the resin, the reaction solution was concentrated and purified with Sephadex LH-20 / DMF (f18 × 400 mm). The obtained peptide was analyzed by ¹ H-NMR (Varian;
Mercury-300) and HPLC (Shima
dzu; SPD-10A, LC-10AT, FCV-1
0AL, DGV-4A, C-R6A).

The sources of purchase of the reagents used above are as follows. Fmoc-D-napAla-OH, Fmoc
-Glu (OtBu) -OH, Fmoc-Pro-O
H, Fmoc-Nle-OH, and Fmoc-Ala
-OH is from Fmoc-Ocg-O from Watanabe Chemical (Hiroshima)
H was purchased from Chiro Tech (USA). PdO (PPh ₃ ) ₄ : tetrakis (triphenylphosphine) palladium was purchased from Aldrich (USA). HATU: azabenzotriazol-1-yl-N,
N, N ', N'-tetramethyluronium tetrafluoroosphate is available from PerSeptive Biosy
purchased from Stems (USA).

Example 6 (Interaction between GS-Nle and pyrenemethanol) About 1 mg of powdered GS-Nle was added to about 5 mL of pH 7.5.
Shake well with phosphate buffer solution of
(Toyo filter paper DISMIC-13 _HP ). The absorbance of this solution at 285 nm becomes about 0.6 (5 × 10 5 assuming a molecular extinction coefficient of 12,000 (285 nm)).
^-5 M). This solution was diluted to a concentration of 1.0 × 10 ^−7.
M. 3 mL of this solution is put into a 1 cm square fluorescent cell and the fluorescence spectrum is measured (excitation wavelength 285 nm,
(Fluorescence wavelength 300-500 nm, slit width, 5 nm (excitation side), 10 nm (fluorescence side)). About 1 mg of powdered pyrenemethanol is shaken well with about 5 mL of a phosphate buffer having a pH of 7.5, and a membrane filter (TOYO filter paper DISMI) is used.
C-13 _HP ). This solution is diluted about 100-fold to make the absorbance at 341 nm 0.346 (concentration of 1.3 × with a molecular extinction coefficient of 27,000 (341 nm)).
10 ^-5 M). 6 μL of this stock solution is added to the cell, and the mixture is stirred for 1 minute with a stirrer in the cell, and then the fluorescence spectrum is measured. This operation was repeated to obtain 325 of the naphthyl group.
The fluorescence intensity in nm is plotted against the number of moles of pyrenemethanol added.

Example 7 (Interaction between GS-Nle and dibutyl phthalate) In the same manner as in Example 6, the fluorescence intensity of the naphthyl group of GS-Nle is plotted against the number of moles of dibutyl phthalate.

[0060]

Industrial Applicability The cyclic peptide of the present invention has the ability to strongly capture lipophilic organic compounds, especially environmental hormones,
In addition, these harmful substances can be selectively captured even in an extremely diluted state, and are useful in environmental measures. In addition, the cyclic peptide of the present invention can specifically capture extremely low concentrations of lipophilic organic compounds in a medium, and can be used for identification or quantification of harmful substances and the like in a medium.

[Brief description of the drawings]

FIG. 1 shows the concept of the cyclic peptide of the present invention.

FIG. 2 shows an outline of a method for producing a cyclic peptide of the present invention using a solid phase.

FIG. 3 shows an outline of a production example of the cyclic peptide of the present invention.

FIG. 4 shows an outline of a production example of the cyclic peptide of the present invention.

FIG. 5 schematically shows the cyclic peptide of the present invention capturing pyrenemethanol, showing the change in the fluorescence intensity.

FIG. 6 is a graph showing the capture coefficient when the cyclic peptide of the present invention captures pyrenemethanol.

FIG. 7 schematically shows the cyclic peptide of the present invention capturing dibutyl phthalate, and shows the change in the capturing coefficient.

Claims (16)

[Claims] 1. The following formula (I): (Wherein, R ¹ and R ² each independently represent a group having a hydrophilic group or a group having a hydrophilic group bonded to a carrier, and X ¹ , X ² , X ³ and X ⁴ are each independently And Z ¹ and Z ² each independently represent a fluorescent group, and n and m each independently represent an integer of 1 to ^3. ) A cyclic peptide or a salt thereof, wherein at least one of the amino acid units constituting the cyclic peptide is a non-natural amino acid unit. 2. The cyclic peptide or a salt thereof according to claim 1, wherein the group having a hydrophilic group is a group containing a carboxyl group, an amino group, a hydroxyl group or a phosphate group. 3. The group having a hydrophilic group is a group represented by the general formula: —R ³ —COOH (wherein, R ³ represents a linear or branched lower alkylene group) or a group thereof. The cyclic peptide according to claim 2, which is a salt, or a salt thereof. 4. The cyclic peptide or a salt thereof according to claim 1, wherein the hydrophobic group is a linear or branched alkyl group, aralkyl group or aryl group. 5. The cyclic peptide or a salt thereof according to claim ^1, wherein Z ¹ and Z ² are an aryl group. 6. R ¹ and R ² are — (CH ₂ ) ₂ —CO
2. The compound according to claim ¹ , which is an OH group, X ¹ , X ² , X ³ and X ⁴ are each independently an alkyl group, Z ¹ and Z ² are a naphthyl group, and n and m are 1. Or the salt thereof. 7. A scavenger for a lipophilic organic compound comprising the cyclic peptide according to claim 1 or a salt thereof. 8. The capturing agent according to claim 7, wherein the lipophilic organic compound is an environmental hormone. 9. The trapping agent according to claim 7, wherein the cyclic peptide according to claim 1 or a salt thereof is bound to a carrier. 10. The capturing agent according to claim 8, wherein the environmental hormone is a phthalic acid ester. 11. A method for capturing and / or recovering a lipophilic organic compound using the capturing agent according to claim 7. 12. The method according to claim 11, wherein the lipophilic organic compound is an environmental hormone. 13. A method for detecting a lipophilic organic compound in a medium using the cyclic peptide according to claim 1 or a salt thereof. 14. The method according to claim 13, wherein the lipophilic organic compound is an environmental hormone. 15. A kit for detecting a lipophilic organic compound in a medium comprising the cyclic peptide according to claim 1 or a salt thereof. 16. The kit according to claim 15, wherein the lipophilic organic compound is an environmental hormone.